GC-MS and LC-MS methods for Flame Retardant (FR) … spectrum of endosulfan (C9H6Cl6O3S, m. wt. 406...
Transcript of GC-MS and LC-MS methods for Flame Retardant (FR) … spectrum of endosulfan (C9H6Cl6O3S, m. wt. 406...
Sicco Brandsma
GC-MS and LC-MS methods for
Flame Retardant (FR) analysis
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Outlines
• Gas Chromatograph
• Different detectors ECD FID NPD and MS
• Focus on Mass spectrometry
• Different GC parameters
• BFRs PBDEs, BDE209, TBBP-A and HBCD
• “New” compounds PFRs, DBDPE, BTBPE, TBPH,
TBB and, PBT
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Gas chromatograph
Flow controller
Carrier gas
Injector port
Chromatogram
Detector
GC column
Temperature programmed
column oven
Multiplier
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GC-Detectors
• Flame Ionisation detector (FID)
• Sensitive for detecting hydrocarbons
• Not really selective measures all flammable compounds
• Electron Capture Detector (ECD)
• Sensitive to halogens and nitro compounds
• Low linear range compared to FID
• Nitrogen Phosphorous Detector (NPD)
• Very sensitive but selective detector that responds almost exclusively to
nitrogen and phosphorous compounds
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ECD chromatogram
• Low selectivity: coeluting compounds can not be separated
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• Electron Impact (EI)
• Hard ionisation fragmentation of the molecular ion
• Library search for EI spectra
• Chemical ionisation (CI)
• Soft ionisation clear spectrum of the Molecular ion
• Use of reagent gas (Methane)
• Positive PCI or negative ECNI mode
• ECNI very sensitive for PBDEs
Mass Spectrometry (1)
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Mass Spectrometry (2)
• MS
• Quadrupole (SIM or SCAN)
• Ion trap
• Time Of Flight (TOF)
• Sector instruments (magnet)
• Tandem Mass spectrometry (MS/MS)
• Two analysers coupled by an collision cell
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327.0145
327.0009
327.0081
Full scan
SIM m/z 327
MS/MS 327 251
HRMS
TCPP TCPP
TCPP
Mass Spectrometry (3)
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• Selection of the Stationary Phase
• Column Dimension
• Temperature Program
• Linear Gas Velocity
• Type of Carrier Gas
GC parameters
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Resolution
R = 0.5
R = 0 R = 1
R = 1.5
R = ΔtR × 2 / (W1 + W2)
R = √N / 4
or
Changing mobile phase composition
Changing column temperature
Changing composition of stationary phase
Changing the column length
• Improving the resolution:
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• The most widely used stationary phases for halogenated pollutants
are: DB-1, DB-5, BPX-5, HT-8, CP-Sil8 CB or CP-Sil 19 CB
polarity scale Stationary phase brand and type names
5 100% Dimethyl polysiloxane ZB-1 MS, CP-Sil 5 CB, DB-1, HP-1 MS, RTX-1
8 5% Phenyl-(arylene)- 95% methyl polysiloxane ZB-5 MS, CP-Sil 8 CB, DB-5 MS, HP-5 MS, RTX-5 MS
17 50% Phenyl-50% methyl polysiloxane ZB-50, DB-17 MS, HP-17, RTX-50
19 14% cyanopropyl-phenyl/86% dimethylpolysiloxane CP-Sil 19 CB
24 75% Phenyl-25% methyl polysiloxane ZB-50, CP-Sil 24 CB
52 polyethylene glycol ZB-WAX, DB-WAX, CP-WAX 52 CB
88 100% 3-cyanopropylpolysiloxane BPX70, CP-Sil 88 CB, RTX-2330
Selection stationary phase
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•Effect of Diameter (ID): Stronger than that of Length
• Length: > 100m: to much pressure required, too long retention
times, < 25: serious loss of separation • ID: <0.10 mm: hardly possible (columns >20m) >0.25 mm: serious loss of separation • Film Thickness: < 0.1 mm: risk for degradation due to poor coating > 0.3 mm: long retention times, risk for tailing peaks
Column Dimensions
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Temperature Program
Temperature program: 4 min, 80oC;
5oC/min to 270; 48 min 270oC.
Temperature program: 4 min, 70oC;
30oC/min to 215oC; 40 min 215oC; 5oC
/min to 270oC; 33 min 270oC
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The Optimum mobile phase velocity
Optimum resolution
The H-u curve is very useful to determine the optimum mobile phase velocity
uopt at which the highest column efficiency will be attained
H = HETP (plate height)
A = Eddy diffusion term
B = Longitudinal diffusion term
u = Linear velocity
C = Resistance to mass transfer coefficient
N = Plate number
L = Column length
Van deemster equation
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N2
He H2
U
H
Selection of carrier gas
•Hydrogen provide optimum resolution at the highest carrier gas velocity; optimum separation within an the sorter time
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PBDEs
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• PBDEs
• GC-EI-MS, GC-ECNI-MS, GC/HRMS or LC-MS/MS
• Advantage and Disadvantage
• EI versus ECNI
• Elution of interfering compounds
• Different GC-columns
• Internal standards
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GC/LR-ECNI-MS GC/LR-EI-MS GC/HRMS LC-MS/MS
LOD 30 fg - 1.7 pg* 0.5 - 32 pg* 0.1 - 4.8 pg* 12 - 30 pg*
Sensitivity ++ - + --
Selectivity No yes yes yes
Labeled standards No (only for BDE209) yes yes yes
Thermal degradation yes yes yes no
Expensive +- - ++ +
Expert training - - ++ +
Libary search No yes yes No*Eljarrat et al, (2002) *Eljarrat et al, (2002) *Alaee et al, (2001) *Abdallah et al, (2009)
J Mass Spectrom 37: 76-84 J Mass Spectrom 37: 76-84 Chemosphere 44: 1489-1495 Anal. Chem., 81, 7460–7467
Advantages and Disadvantages
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Mass spectrum of endosulfan (C9H6Cl6O3S, m. wt. 406 g/mole, CAS number 115-29-7) in EI
(upper panel) and ECNI (lower panel). EI produces extensive fragmentation with very little molecular
ion formed (M+), but the molecular anion (M–) is the most abundant peak in ECNI. The fragment at 372
m/z is formed by loss of chlorine which also appears at 35 and 37 m/z.
Agilent Technologies
GC-EI MS versus GC-ECNI MS
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Interfering compounds
•PBBs: BB-153 may interfere with
BDE-154 when ECNI-MS is applied.
•TBBPA may interfere with BDE-153
when ECNI-MS is applied.
•MeO-PBDEs: 5-Cl-6-MeO-BDE47
and 6’-Cl-2’-MeO-BDE68 may
interfere with BDE-99 when ECNI-
MS is applied.
•DiMeO-PBDE: 2’,6-DiMeO-BDE68
may interfere with BDE-99 when
ECNI-MS is applied.
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This database is useful for determining the most suitable column for quantitative,
congener-specific PBDE analysis.
Resolving power of seven different GC- columns
Korytár et al,(2005) J Chromatogr A 1065:239–249
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15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00
Time-->
BDE190
54.1
BB169
49.4
BDE138
46.1
HBCD
45.4
TBBP-A+
BDE153
43.8
BB153+BDE154
42.3
me-TBBP-A
42.2
BDE85
41.2
BDE99
39.1
BDE119
38.3
BDE100
37.8
BB101
35.8
BDE77
35.3
BDE66
33.6
BDE47
32.6
BDE71
31.5
BDE75
30.5
BB49
25.3
BB52
24.2
BDE28
21.2
Internal standard
CB112
17.5
BB1
5
13.8
CP-Sil8 CB MS 50 m x 0.25 mm x 0.25 µm
GC-ECNI MS chromatogram
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Internal standards
•Internal std for GC-ECNI-MS
• BDE58
• BDE77
• BDE128
• 13C12 BDE209
• F-PBDEs
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Conclusions
• GC-ECNI-MS is the best all-round method to
analyze PBDEs
• Be aware of coeluting of “new” brominated
compounds (only measuring m/z 79 and 81 in
ECNI mode)
• However when higher detection limits are not a
problem GC-EI-MS or LC-MS/MS are good
alternatives
• Advantage of EI over ECNI mode is the use of
labelled internal standards
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BDE209
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BDE209
BDE206
BDE207
BDE208
DB-5, 15 m x 0.20 mm x 0.2 µm
Pulsed splitless injection
degradation
of decaBDE
>15m
BDE209 GC/ECNI-MS
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Loss of response of higher brominated PBDE
due to matrix
decaBDE
Matrix effects
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MS spectra (ECNI) of BDE209
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492
496 498
500
13C12 decaBDE
482
484
486 488
490
494
492
496
MS spectra (ECNI) of 13C12 BDE209
Bjorklund et al, 2003 J. Mass Spectrom. 38, 394-400
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200°C
250°C
300°C
15.00 15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.50 22.00
Ion source (ECNI) temperature
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• Use 13C decaBDE as internal standard
• Use <15 m GC column
• Use short injector residence times (pulsed splitless) or
on-column injection
• Reduce sample exposure to glassware and reduce if
possible number of pieces of glassware used
• If possible physically segregate sample by type and
analysis in separate areas
• Reduce UV-light exposure (UV-filters or amber
glassware)
• Reduce and avoid dust
Summary for BDE209
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HBCD GC or LC ?
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15.00 20.00 25.00 30.00 35.00 40.00 45.00
2000 4000 6000 8000
10000 12000 14000 16000 18000 20000 22000 24000 26000 28000 30000 32000 34000 36000 38000 40000 42000 44000 46000 48000 50000
Rt (min)
Abundance
GC-MS (ECNI) HBCD (I)
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15.00 20.00 25.00 30.00 35.00 40.00 45.00
500
1000
1500
2000
2500
3000
3500
Rt (min)
Abundance
TetraBCDe
PentaBCDe
HBCD
GC-MS (ECNI) HBCD (II)
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m/z 79
28 75
71
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HBCD
15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 0
500
1000
1500
2000
2500
3000
Rt (min)
Abundance
47
58
66
77
100
119
99
85 Dime
-TBBP
-A
154
153 138
183 190
GC-MS (ECNI) HBCD and PBDEs
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32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
55000
60000
65000
Rt (min)
Abundance
BD
E1
53
BD
E1
54
BD
E8
5 B
DE
99
BD
E1
00
BD
E6
6
BD
E5
8
BD
E4
7
BD
E4
9
HBCD
Sediment sample, Scheldt estuary
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43.00 43.20 43.40 43.60 43.80 44.00 44.20 44.40 44.60 44.80 0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
55000
Rt (min)
Abundance α-HBCD
β-HBCD
γ-HBCD
GC-MS ECNI response HBCD isomers
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4x10
0
1
2
3
4
Abundance vs. Acquisition Time (min)1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
- MRM (652.7 -> 79.0) paling 1-1 met water gradient 1.d
1 1
4x10
0
1
2
3
4
- MRM (652.7 -> 81.0) paling 1-1 met water gradient 1.d
1 1
3x10
0
2
4
- MRM (652.7 -> 652.7) paling 1-1 met water gradient 1.d
1 1
3x10
0
2
4
6
- MRM (640.7 -> 79.0) paling 1-1 met water gradient 1.d
1 1
3x10
0
2
4
6
- MRM (640.7 -> 81.0) paling 1-1 met water gradient 1.d
1 1
3x10
0
0.5
1
- MRM (640.7 -> 640.7) paling 1-1 met water gradient 1.d
1 1
α β
γ 13C-labeled
13C-labeled
13C-labeled
Eel, LC-MS/MS, HBCD
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1x10
0
1
2
3
Abundance vs. Acquisition Time (min)3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8 8 8.2 8.4 8.6 8.8 9 9.2 9.4 9.6 9.8 10 10.2 10.4 10.6 10.8 11 11.2 11.4 11.6 11.8 12 12.2 12.4 12.6 12.8
- MRM (640.7 -> 79.0) mengstandaard 1 ngml 6 ul injectie voor Pim 001.d
7.575
6.8384.343 5.674 7.307
6.4895.135 7.0113.862
1 1 2 2 3 3
1x10
0
1
2
- MRM (640.7 -> 81.0) mengstandaard 1 ngml 6 ul injectie voor Pim 001.d
7.548
4.6223.394 7.6947.1354.432 5.172 5.844 6.163 6.7333.679
1 1 2 2 3 3
1x10
0
0.5
1
- MRM (640.7 -> 79.0) mengstandaard 1 ngml 6 ul injectie voor Pim 001.d
1 1 2 2 3 3
1x10
0
0.5
1
- MRM (640.7 -> 81.0) mengstandaard 1 ngml 6 ul injectie voor Pim 001.d
1 1 2 2 3 3
1x10
0
2
4
6
- MRM (640.7 -> 79.0) mengstandaard 1 ngml 6 ul injectie voor Pim 001.d
9.378
10.8368.663 9.601 9.877 11.4479.044 12.64810.271 11.742
1 1 2 2 3 3
1x10
0
2
4
- MRM (640.7 -> 81.0) mengstandaard 1 ngml 6 ul injectie voor Pim 001.d
9.389
8.838 9.922 11.28310.512 12.699
1 1 2 2 3 3
Zorbax Eclips, 2.1 x 150 mm x 3.5 um
6 pg
2x10
0
1
2
Abundance vs. Acquisition Time (min)0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7
- MRM (640.3 -> 79.0) HBCD0012.d
0.725
0.261 1.1440.835
1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8
2x10
0
1
2
- MRM (640.3 -> 81.0) HBCD0012.d
0.7151 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8
2x10
0
1
2
3
- MRM (640.3 -> 79.0) HBCD0012.d
1.5131 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8
2x10
0
0.5
1
1.5
2
- MRM (640.3 -> 81.0) HBCD0012.d
1.5061 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8
2x10
0
1
2
- MRM (640.3 -> 79.0) HBCD0012.d
2.292
2.177
1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8
2x10
0
0.5
1
1.5
2
- MRM (640.3 -> 81.0) HBCD0012.d
2.3091 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8
Zorbax SB-C18, 2.1 x 30 mm x 3.5 um
6 pg
Normal LC vs. Rapid Resolution LC columns
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0
50
100
150
200
250
0 50 100 150 200 250
LC-MS
GC
-MS
-GC: internal standard conc. (no 13C)
-GC: response factors isomers differ
-isomers transferred into each other
-LC: Ion-suppression
Quantification of HBCD: LC vs. GC
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Tomy et al. 2005, Rapid Comm. Mass Spectr. 19, 2819-2826
Uncorrected Corrected, 13C-labelled
LC-MS/MS Ion suppression of HBCD signal by matrix compound
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HBCD • GC: Thermal degradation on GC column • Thermal degradation causes interfering peaks with PBDEs • LC-MS preferred method • Use of 13C-labeled IS is preferred • LC-MS/MS (1 pg) as sensitive as GC-MS (0.5 pg)
Conclusions
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TBBP-A GC or LC ?
44
GC-MS TBBP-A
• Without derivatization poor calibration curves
• Interference TBBP-A and BDE153
• With derivatization -> dime-TBBP-A
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GC-MS TBBP-A
43.60 43.80 44.00 44.20 44.40 44.60 44.80 45.00 45.20 45.40 45.60 45.80
TBBP-A standard 10 ng/ml, 1 µl injection
43.60 43.80 44.00 44.20 44.40 44.60 44.80 45.00 45.20 45.40
Sediment +10 ng/ml TBBP-A
m/z 543
m/z 543
Sediment, Scheldt estuary
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44.00 44.20 44.40 44.60 44.80 45.00 45.20 45.40 0 50
100 150 200 250 300 350 400 450 500 550 600 650 700 750 800
Abundance 44.00 44.20 44.40 44.60 44.80 45.00 45.20 45.40 45 50 55 60 65 70 75 80 85 90 95
100 105 110 115 120 125 130 135 140
Abundance TBBP-A m/z 543
BDE153+TBBP-A m/z 79
TBBP-A and BDE153
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0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 Rt (min)
TBBP-A
13C-TBBP-A
m/z 543
m/z 555
500 510 520 530 540 550 560 570 580 590 600 610
m/z
543 555
541 553 545
557
539 547 559
TBBP-A 13C-TBBP-A
• LC-MS easy to perform
TBBP-A analysis
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0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 Time 0
100
%
0
100
%
C143350 6: SIR of 1 Channel ES-
640.50
6.15e4
Area
30.92
28.73
C143350 3: SIR of 1 Channel ES-
542.90
1.65e6
Area
23.01
a-HBCD g-HBCD
B-HBCD
TBBP-A
LC-MS/MS: HBCD and TBBP-A
49
TBBP-A
• LC-MS preferred method, use 13C-labelled IS
• GC-MS (ECNI) BDE153 and TBBP-A interfere
• 13C-TBBP-A can cause interference with BDE153
• Sample treatment most critical step
Conclusions
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PFRs
51
PFRs
TiBP TBP TCEP TCPP
TDCPP TBEP TPP EHDP
TEHP TCP DBPhP DPhBP
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Compound Acronym Retention time Precursor Ion Product Ion Fragmentor Collision Energy
tri-n-butyl phosphate D27 * TBP D27 15,8 294.4 166.1 100 15
294.4 102.1 100 15
tri-n-butyl phosphate TBP 15,6 267.2 155.1 100 10
267.2 99.1 100 10
tris(2-chloroethyl) phosphate TCEP 6,4 287 99.1 100 19
TCEP
285 63.1 110 18
tris-iso-butyl phosphate TiBP 15,8 267.2 155.1 75 8
267.2 99.1 75 8
tris(chloro 2-propyl) phosphate TCPP 11,6 329 99 100 15
tris(1,3-dichloro-2-propyl)phosphate TDCPP 14,4 433 99.4 125 30
tris(2-butoxyethyl) phosphate TBEP 16,4 399.4 299.2 125 15
399.4 199.1 125 15
tris(2-ethylhexyl) phosphate TEHP 22,8 435.4 113.3 125 10
435.4 99.1 125 10
triphenyl phosphate d15 * TPP d15 16,4 342.2 160.2 175 40
342.2 82.1 175 40
triphenyl phosphate TPP 14,5 327.2 152.1 175 35
327.2 77.1 175 35
tricresyl phosphate TCP 17,1 369.2 165.2 175 35
369.2 91.1 175 35
2-Ethylhexyl diphenyl phosphate EHDP 17,5 251.1 152.2 150 28
251.1 77.2 150 28
Butyl diphenyl phosphate BDphP 15 307.2 251.1 100 6
Dibutyl phenyl phosphate DBphP 15,4 287.2 175.1 100 8
HPLC column 150 x 3 mm Luna C18 (2) 3 um column (Phenomenex)
Acronyms, Rt and MRM for twelve PFRs
53
GC/EI-MS
Van den eede et al, (2011) Environment International 37 454–461
54
PFRs in Belgian home dust (n=33) μg/g
Van den eede et al, (2011) Environment International 37 454–461
55
2100 5400
ng/g
lw
/TO
C
ng/g
lw
/TO
C
Pelagic food web Benthic food web
Pelagic and Benthic food web
56
Conclusion GC or LC • LC-MS/MS is more selective • Use of labelled Std • Less interference of matrices (biota) GC/EI-MS • Good method for analyzing dust (less matrices) • No ion suppression • Separation of isomers (TCPP and TCP)
57
TBB and TBPH
BTBPE and DBDPE
58 Ali et al., (2011) Anal. Bioanal. Chem., 400, 3073-3083
59
GC/ECNI-MS chromatograms revealing the relative retention times of the
primary BDE congeners, TBB and TBPH on a 15 m DB5-MS column
Stapleton et al, (2008)Environ. Sci. Technol. 42, 6910–6916
GC/ECNI-MS chromatogram
60
TBB was quantified using ion fragment (m/z) 357 (Quant)and 471 (Qual)
TBPH was quantified using ionfragments (m/z) 463 (Quant) and 515 (Qual)
Stapleton et al, (2008)Environ. Sci. Technol. 42, 6910–6916
Full scan spectra of TBB and TBPH
61
GC/ECNI-MS chromatogram (m/z 79-+81-) of an standard solution
Kierkegaard et al, (2004) Environ. Sci. Technol., 38, 3247-3253
DBDPE first detected in the environment
62
Ali et al., (2011) Anal. Bioanal. Chem., 400, 3073-3083
DBDPE and BTBPE
63 Ali et al., (2011) Anal. Bioanal. Chem., 400, 3073-3083
How to separate HCDBCO from TBB
64
Concentrations observed in Dust
65
PBT
GC/ECNI-MS
m/z 79, 81
66
Review about Novel BFRs
67
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